Self-containing lactococcus strain

ABSTRACT

The invention relates to a recombinant  Lactococcus  strain, with environmentally limited growth and viability. More particularly, it relates to a recombinant  Lactococcus  that can only survive in a medium, where well-defined medium compounds are present. A preferred embodiment is a  Lactococcus  that may only survive in a host organism, where such medium compounds are present, but cannot survive outside the host organism in the absence of such medium compounds.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/687,996, filed Oct. 17, 2003, pending, which is a continuation of PCTInternational Patent Application No. PCT/EP02/04942, filed on May 3,2002, designating the United States of America, and published, inEnglish, as PCT International Publication No. WO 02/090551 A2 on Nov.14, 2002, the contents of the entirety of which are incorporated hereinby this reference.

TECHNICAL FIELD

The invention relates to a recombinant Lactococcus strain, withenvironmentally limited growth and viability. More particularly, itrelates to a recombinant Lactococcus that can only survive in a medium,where well-defined medium compounds are present. A preferred embodimentis a Lactococcus that may only survive in a host organism, where themedium compounds are present, but cannot survive outside the hostorganism in an absence of the medium compounds. Moreover, theLactococcus can be transformed with prophylactic and/or therapeuticmolecules and can, as such, be used to treat diseases such asinflammatory bowel diseases.

BACKGROUND

Lactic acid bacteria have long been used in a wide variety of industrialfermentation processes. They have generally-regarded-as-safe (“GRAS”)status, making them potentially useful organisms for the production ofcommercially important proteins. Indeed, several heterologous proteins,such as Interleukin-2, have been successfully produced in Lactococcusspp (Steidler et al., 1995). It is, however, undesirable that suchgenetically modified microorganisms survive and spread into theenvironment.

To avoid unintentional release of genetically modified microorganisms,special guidelines for safe handling and technical requirements forphysical containment are used. Although this may be useful in industrialfermentations, the physical containment is generally considered asinsufficient, and additional biological containment measures are takento reduce the possibility of survival of the genetically modifiedmicroorganism in the environment.

Biological containment is extremely important in cases where physicalcontainment is difficult or even inapplicable. This is, amongst others,the case in applications where genetically modified microorganisms areused as live vaccines or as a vehicle for delivery of therapeuticcompounds. Such applications have been described, for example, in PCTInternational Publication Number WO 97/14806, which discloses thedelivery of biologically active peptides, such as cytokines, to asubject by recombinant noninvasive or nonpathogenic bacteria. Further,PCT International Publication Number WO 96/11277 describes the deliveryof therapeutic compounds to an animal or human by administering arecombinant bacterium encoding a therapeutic protein. Steidler et al.(2000) describe the treatment of colitis by administration of arecombinant Lactococcus lactis secreting Interleukin-10. Such a deliverymay indeed be extremely useful to treat a disease in an affected humanor animal, but the recombinant bacterium may act as a harmful andpathogenic microorganism when it enters a nonaffected subject, and anefficient biological containment that avoids such unintentionalspreading of the microorganism is needed.

Biological containment systems for host organisms may be passive andbased on a strict requirement of the host for a specific growth factoror a nutrient that is not present or is present in low concentrations inthe outside environment. Alternatively, it may be active and, based onso-called suicidal genetic elements in the host, wherein the host iskilled in the outside environment by a cell-killing function, encoded bya gene that is under the control of a promoter only being expressedunder specific environmental conditions.

Passive biological containment systems are well known in microorganismssuch as Escherichia coli or Saccharomyces cerevisiae. Such E. colistrains are disclosed, for example, in U.S. Pat. No. 4,190,495. Also,PCT International Publication Number WO 95/10621 discloses lactic acidbacterial suppressor mutants and their use as means of containment inlactic acid bacteria, but in that case, the containment is on theplasmid level, rather than on the level of the host strain and itstabilizes the plasmid in the host strain, but does not providecontainment for the genetically modified host strain itself.

Active suicidal systems have been described by several authors. Suchsystems consist of two elements: a lethal gene and a control sequencethat switches on the expression of the lethal gene under nonpermissiveconditions. For example, PCT International Publication Number WO95/10614 discloses the use of a cytoplasmatically active truncatedand/or mutated Staphylococcus aureus nuclease as a lethal gene. PCTInternational Publication Number WO 96/40947 discloses a recombinantbacterial system with environmentally limited viability, based on theexpression of either an essential gene, expressed when the cell is inthe permissive environment and not expressed or temporarily expressedwhen the cell is in the nonpermissive environment, and/or a lethal gene,wherein expression of the gene is lethal to the cell and the lethal geneis expressed when the cell is in the nonpermissive environment but notwhen the cell is in the permissive environment. PCT InternationalPublication Number WO 99/58652 describes a biological containment systembased on the relE cytotoxin. However, most systems have been elaboratedfor Escherichia coli (Tedkin et al., 1995; Knudsen et al., 1995;Schweder et al., 1995) or for Pseudomonas (Kaplan et al., 1999; Molinoet al., 1998). Although several of the containment systems theoreticallycan by applied to lactic acid bacteria, no specific biologicalcontainment system for Lactococcus has been described that allows theusage of a self-containing and transformed Lactococcus to deliverprophylactic and/or therapeutic molecules in order to prevent and/ortreat diseases.

DISCLOSURE OF THE INVENTION

The invention includes a suitable biological containment system forLactococcus. A first aspect of the invention is an isolated strain ofLactococcus sp. comprising a defective thymidylate synthase gene.

Another aspect of the invention is the use of a strain according to theinvention as a host strain for transformation, wherein the transformingplasmid does not comprise an intact thymidylate synthase gene.

Still another aspect of the invention is a transformed strain ofLactococcus sp. according to the invention, comprising a plasmid thatdoes not comprise an intact thymidylate synthase gene. Another aspect ofthe invention relates to a transformed strain of Lactococcus sp.comprising a gene or expression unit encoding a prophylactic and/ortherapeutic molecule such as Interleukin-10. Consequently, the presentinvention also relates to the usage of a transformed strain ofLactococcus sp. to deliver prophylactic and/or therapeutic moleculesand, as such, to treat diseases. Methods to deliver the molecules andmethods to treat diseases such as inflammatory bowel diseases areexplained in detail in PCT International Publication Numbers WO 97/14806and WO 00/23471 to Steidler et al. and in Steidler et al. (Science 2000,289: 1352), the contents of all of which are incorporated herein by thisreference.

Another aspect of the invention is a medical preparation comprising atransformed strain of Lactococcus sp., according to the invention.

The invention further demonstrates that the transformed strainssurprisingly pass the gut at the same speed as the control strains,showing that their loss of viability indeed is not different from thatof the control strains. However, once the strain is secreted in theenvironment, for example, in the feces, it is not able to survive anylonger.

The transforming plasmid can be any plasmid, as long as it does notcomplement the thyA mutation. It may be a self-replicating plasmid thatpreferably carries one or more genes of interest and one or moreresistance markers, or it may be an integrative plasmid. In the lattercase, the integrative plasmid itself may be used to create the mutationby causing integration at the thyA site, whereby the thyA gene isinactivated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Map of the MG1363 thyA locus.

FIG. 2: Schematic representation of the different expression modules aspresent on pOThy plasmids ands genomic integrants of hIL-10. Black partsrepresent original L. lactis MG1363 genetic information; white partsrepresent recombinant genetic information.

FIGS. 3A and 3B: PCR identification of Thy11 (Thy11 1.1 and Thy11 7.1represent individually obtained, identical clones). Standard PCRreactions were performed by using aliquots of saturated cultures of theindicated strains as a source of a DNA template. FIG. 3A shows anagarose gel of the products of the indicated PCR reactions. FIG. 3Bshows the positions at which primers attach in the thyA (1), upstream(2) or downstream (3) PCR's. Oligonucleotide primers used:

(1): ATgACTTACgCAgATCAAgTTTTT (SEQ ID NO: 8 of theaccompanying SEQUENCE LISTING, which isincorporated herein by this reference and (SEQ ID NO: 9)TTAAATTgCTAAATCAAATTTCAATTg (2): (SEQ ID NO: 10) TCTgATTgAgTACCTTgACCand (SEQ ID NO: 11) gCAATCATAATTggTTTTATTg (3): (SEQ ID NO: 12)CTTACATgACTATgAAAATCCg and (SEQ ID NO: 13) cTTTTTTATTATTAgggAAAgCA.

FIGS. 4A and 4B: PCR identification of Thy11, Thy12, Thy15 and Thy16.Standard PCR reactions were performed by using three-day old colonies ofthe indicated strains as a source of DNA template. FIG. 4A shows thepositions at which primers attach in the upstream (1), downstream (2) orthyA (3), PCRs. Oligonucleotide primers used:

(1): ATgACTTACgCAgATCAAgTTTTT (SEQ ID NO: 8) andTTAAATTgCTAAATCAAATTTCAATTg (SEQ ID NO: 9) (2): TCTgATTgAgTACCTTgACC(SEQ ID NO: 10) and gCAATCATAATTggTTTTATTg (SEQ ID NO: 11) (3):CTTACATgACTATgAAAATCCg (SEQ ID NO: 12) and cTTTTTTATTATTAgggAAAgCA.(SEQ ID NO: 13)FIG. 4B shows an agarose gel of the products of the indicated PCRreactions.

FIGS. 5A and 5B: Southern blot analysis of the indicated strains.Chromosomal DNA was extracted and digested with the indicatedrestriction enzymes. Following agarose gel electrophoresis, the DNA wastransferred to a membrane and the chromosome structure around the thyAlocus was revealed by use of DIG-labeled thyA or hIL-10 DNA fragments(FIG. 5A). FIG. 5B shows a schematic overview of the predicted structureof the thyA locus in both MG1363 and Thy11.

FIG. 6A shows a schematic overview of part of the predicted structure ofthe L. lactis chromosome at the thyA locus in MG1363, Thy11, Thy12,Thy15 and Thy16. Numbers indicate base pairs. FIG. 6B illustrates aSouthern blot analysis of the indicated strains. Chromosomal DNA wasextracted and digested with NdeI and SpeI restriction enzymes. Followingagarose gel electrophoresis, the DNA was transferred to a membrane andthe chromosome structure around the thyA locus was revealed by use ofDIG-labeled thyA or hIL-10 DNA fragments.

FIGS. 7A and 7B: Production of hIL-10. FIG. 7A shows a western blotrevealed with anti-hIL-10 antiserum of culture supernatant andcell-associated proteins of the indicated strains. FIG. 7B showsquantification (by ELISA) of hIL-10 present in the culture supernatant.

FIGS. 8A and 8B: Production of hIL-10. FIG. 8A shows quantification (byELISA) of hIL-10 present in the culture supernatant of the indicatedstrains. FIG. 8B shows a western blot revealed with anti-hIL-10antiserum of culture supernatant proteins of the indicated strains.

FIG. 9: Production of hIL-10 by the L. lactis strains LL108 carryingpOThy11, pOThy12, or pOThy16. Quantification (by ELISA) of hIL-10present in the culture supernatant of the indicated strains is shown.The N-terminal protein sequence of the recombinant hIL-10 was determinedby Edman degradation and was shown to be identical to the structure aspredicted for the mature, recombinant hIL-10. The protein showed fullbiological activity.

FIGS. 10A and 10B: Growth rate of the indicated strains in GM17containing 100 μg/ml (T100), 50 μg/ml (T50), 25 μg/ml (T25), or no (T0)extra thymidine and possibly supplemented with 5 μg/ml of erythromycin(E). Saturated overnight cultures (prepared in T50) were diluted 1:100in the indicated culture media. FIG. 10A shows the kinetics ofabsorbance accumulation. FIG. 10B shows the kinetics of the number ofcolony-forming units (cfu) per ml of culture.

FIG. 11: Growth rate of MG1363 and Thy12 in thymidine-free medium (TFM).TFM was prepared by growing L. lactis Thy12 bacteria in GM17, removingthe bacteria by subsequent centrifugation and filtration on a 0.22 μmpore size filter, adjusting the pH to 7.0 and autoclaving. MG1363 andThy12 bacteria were collected from an overnight culture in GM17 orGM17+50 μg/ml of thymidine, respectively, and washed in M9 buffer (6Na₂HPO₄, 3 g/l KH₂PO₄, 1 g/l NH₄Cl, 0.5 g/l NaCl in water). Thesuspensions of both were either diluted in TFM or TFM supplemented with50 μg/ml of thymidine (T50). CFU counts were determined at differenttime points: t=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 20 hours. This showsthat Thy12 viability is severely impaired in the absence of thymidine.

FIG. 12: Intestinal passage and viability: L. lactis MG1363 wastransformed with the plasmid pLET2N, which carries a chloramphenicol(Cm) resistance marker. L. lactis Thy12 was transformed with the plasmidpT1NX, which carries an erythromycin (Em) resistance marker. Of bothstrains, 10⁹ bacteria were resuspended in BM9 (6 g/l Na₂HPO₄, 3 g/lKH₂PO₄, 1 g/l NH₄Cl, 0.5 g/l NaCl in 25 mM NaHCO₃+25 mM Na₂CO₃), mixedand inoculated in three mice at t=0h. Feces were collected at the timeintervals −1 to 0, 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to7, 7 to 8, 8 to 9, 9 to 10 and 10 to overnight. All samples wereresuspended in isotonic buffer and appropriate dilutions were plated onGM17 (M17 medium, Difco, St. Louis, Mo., supplemented with 0.5% glucose)plates containing either Cm, Em or Em+50 μg/ml thymidine. Colony-formingunits for the different plates are represented in the graph.

DETAILED DESCRIPTION OF THE INVENTION

As previously identified, the invention includes a suitable biologicalcontainment system for Lactococcus. In one aspect, the invention is anisolated strain of Lactococcus sp. comprising a defective thymidylatesynthase gene. Preferably, the defective thymidylate synthase gene isinactivated by gene disruption. Even more preferably, the Lactococcussp. is Lactococcus lactis. A special embodiment is a Lactococcus sp.strain, preferably Lactococcus lactis, more preferably a Lactococcuslactis MG1363 derivative, wherein the thymidylate synthase gene has beendisrupted and replaced by an Interleukin-10 expression unit. TheInterleukin-10 expression unit is preferably, but not limited to, ahuman Interleukin-10 expression unit or gene encoding for humanInterleukin-10.

The Lactococcus lactis subsp. lactis thymidylate synthase gene (thyA)has been cloned by Ross et al. (1990a). Its sequence is comprised in SEQID NO:3 and SEQ ID NO:5. European Patent Application Publication Number0406003 discloses a vector devoid of antibiotic resistance and bearing athymidylate synthase gene as a selection marker; the same vector hasbeen described by Ross et al. (1990b). However, this vector could not beused in a Lactococcus lactis strain due to the lack of a suitable thyAmutant that has never been described. The present invention discloseshow to construct such a mutant by gene disruption using homologousrecombination in Lactococcus. In a preferred embodiment, the thyA geneis disrupted by a functional human Interleukin-10 expression cassette.However, it is clear that any construct can be used for gene disruption,as long as it results in an inactivation of the thyA gene or in aninactive thymidylate synthase. As a nonlimiting example, the homologousrecombination may result in a deletion of the gene, in one or more aminoacid substitutions that lead to an inactive form of the thymidylatesynthase, or in a frame shift mutation resulting in a truncated form ofthe protein.

Such a Lactococcus sp. thyA mutant is very useful as a host strain fortransformation in situations where more severe containment than purelyphysical containment is needed. Indeed, thyA mutants cannot survive inan environment without or with only a limited concentration of thymidineand/or thymine. When such a strain is transformed with a plasmid thatdoes not comprise an intact thyA gene and cannot complement themutation, the transformed strain will become suicidal in athymidine/thymine-poor environment. Such a strain can be used in afermentor as an additional protection for the physical containment.Moreover, the present invention discloses that such a strain isespecially useful in cases where the strain is used as a deliveryvehicle in an animal body. Indeed, when such a transformed strain isgiven, for example, orally to an animal—including humans—it survives inthe gut, provided that a sufficiently high concentration ofthymidine/thymine is present, and produces homologous and/orheterologous proteins, such as human Interleukin-10, that may bebeneficial for the animal.

The invention further demonstrates that the transformed strainssurprisingly pass the gut at the same speed as the control strains,showing that their loss of viability indeed is not different from thatof the control strains. However, once the strain is secreted in theenvironment, for example, in the feces, it is not able to survive anylonger.

The transforming plasmid can be any plasmid, as long as it does notcomplement the thyA mutation. It may be a self-replicating plasmid thatpreferably carries one or more genes of interest and one or moreresistance markers, or it may be an integrative plasmid. In the lattercase, the integrative plasmid itself may be used to create the mutationby causing integration at the thyA site, whereby the thyA gene isinactivated. Preferably, the active thyA gene is replaced by doublehomologous recombination by a cassette comprising the gene or genes ofinterest, flanked by targeting sequences that target the insertion tothe thyA target site. It is of extreme importance that these sequencesare sufficiently long and sufficiently homologous to integrate thesequence into the target site. Preferably, the targeting sequencesinclude at least 100 contiguous nucleotides of SEQ ID NO:1 at one sideof the gene of interest and at least 100 contiguous nucleotides of SEQID NO:2 at the other side. More preferably, the targeting sequencesconsist of at least 500 contiguous nucleotides of SEQ ID NO:1 at oneside of the gene of interest and at least 500 contiguous nucleotides ofSEQ ID NO:2 at the other side. Most preferably, the targeting sequencesconsist of SEQ ID NO:1 at one side of the gene of interest and SEQ IDNO:2 at the other side, or the targeting sequences consist of at least100 nucleotides that are at least 80% identical, preferably 90%identical to a region of SEQ ID NO:1 at one side of the gene of interestand at least 100 nucleotides that are at least 80% identical, preferably90% identical to a region of SEQ ID NO:2 at the other side of the geneof interest. Preferably, the targeting sequences consist of at least 500nucleotides that are at least 80% identical, preferably 90% identical toa region of SEQ ID NO:1 at one side of the gene of interest and at least500 nucleotides that are at least 80% identical, preferably 90%identical to a region of SEQ ID NO:2 at the other side of the gene ofinterest. Most preferably, the targeting sequences consist of at least1000 nucleotides that are at least 80% identical, preferably 90%identical to a region of SEQ ID NO:1 at one side of the gene of interestand at least 1000 nucleotides that are at least 80% identical,preferably 90% identical to a region of SEQ ID NO:2 at the other side ofthe gene of interest. The percentage identity is measured with BLAST,according to Altschul et al. (1997). A preferred example of a sequencehomologous to SEQ ID NO:1 is given in SEQ ID NO:7. For the purpose ofthe invention, SEQ ID NO:1 and SEQ ID NO:7 are interchangeable.

Transformation methods of Lactococcus are known to the person skilled inthe art and include, but are not limited to, protoplast transformationand electroporation.

A transformed Lactococcus sp. strain according to the invention isuseful for the delivery of prophylactic and/or therapeutic molecules andcan be used in a pharmaceutical composition. The delivery of suchmolecules has been disclosed, as a nonlimiting example, in PCTInternational Publication Numbers WO 97/14806 and WO 98/31786.Prophylactic and/or therapeutic molecules include, but are not limitedto, polypeptides such as insulin, growth hormone, prolactin, calcitonin,group 1 cytokines, group 2 cytokines and group 3 cytokines andpolysaccharides such as polysaccharide antigens from pathogenicbacteria. A preferred embodiment is the use of a Lactococcus sp. strainaccording to the invention to deliver human Interleukin-10. This straincan be used in the manufacture of a medicament to treat Crohn's diseaseas indicated herein.

The invention is further explained with the use of the followingillustrative examples.

EXAMPLES

From L. lactis MG1363 (Gasson, 1983) regions flanking the sequenceaccording to Ross et al. (1990a) have been cloned.

The knowledge of these sequences is of critical importance for thegenetic engineering of any Lactococcus strain in a way as describedbelow, as the strategy will employ double homologous recombination inthe areas of 1000 by at the 5′ end (SEQ ID NO:1) and 1000 by at the 3′end (SEQ ID NO:2) of thyA, the “thyA target.” These sequences are notavailable from any public source to date. These flanking DNA fragmentshave been cloned and their sequence has been identified. The sequence ofthe whole locus is shown in SEQ ID NO:3; a mutant version of thissequence is shown in SEQ ID NO:5. Both the 5′ and 3′ sequences aredifferent from the sequence at GenBank AE006385 describing the L. lactisIL1403 sequence (Bolotin, in press) or at AF336368 describing the L.lactis subsp. lactis CHCC373 sequence. From the literature, it isapparent that homologous recombination by use of the published sequencesadjacent to thyA (Ross et al., 1990a) (86 by at the 5′ end and 31 by atthe 3′ end) is virtually impossible due to the shortness of thesequences. Indeed, Biswas et al. (1993) describe a logarithmicallydecreasing correlation between the length of the homologous sequencesand the frequency of integration. The sequences of L. lactis Thy 11, Thy12, Thy 15 and Thy 16 at the thyA locus as determined in the presentinvention are given by SEQ ID NOS:19, 20, 21, 22 respectively.

The thyA replacement is performed by making suitable replacements in aplasmid-borne version of the thyA target, as described below. Thecarrier plasmid is a derivative of pORI19 (Law et al., 1995), areplication-defective plasmid, which only transfers the erythromycinresistance to a given strain when a first homologous recombination, ateither the 5′ 1000 by or at the 3′ 1000 by of the thyA target. A secondhomologous recombination at the 3′ 1000 by or at the 5′ 1000 by of thethyA target yields the desired strain.

The thyA gene is replaced by a synthetic gene encoding a protein whichhas the L. lactis Usp45 secretion leader (van Asseldonk et al., 1990)fused to a protein of an identical amino acid sequence when: (a) themature part of human-Interleukin 10 (hIL-10) or (b) the mature part ofhIL-10 in which proline at position 2 has been replaced with alanine or(c) the mature part of hIL-10 in which the first two amino acids havebeen deleted; (a), (b) and (c) are called hIL-10 analogs, the fusionproducts are called Usp45-hIL-10.

The thyA gene is replaced by an expression unit comprising thelactococcal P1 promoter (Waterfield et al., 1995), the E. colibacteriophage T7 expression signals, putative RNA stabilizing sequenceand modified gene10 ribosomal binding site (Wells and Schofield, 1996).

At the 5′ end, the insertion is performed in such way that the ATG ofthyA is fused to the P1-T7Usp45-hIL-10 expression unit.

(SEQ ID NO: 27) 5′ agataggaaaatttc atg acttacgcagatcaagttttt . . .thyA wild-type (SEQ ID NO: 14)gattaagtcatcttacctctt . . . P1-T7-usp45-hIL10 (SEQ ID NO: 15) 5′agataggaaaatttc atg gattaagtcatcttacctctt . . . thyA, P1-T7-usp45-hIL10

Alternatively, at the 5′ end, the insertion is performed in such a waythat the thyA ATG is not included:

(SEQ ID NO: 28) 5′ agataggaaaatttcacttacgcagatcaagttttt . . .thyA wild-type (SEQ ID NO: 14)gattaagtcatcttacctctt . . . P1-T7-usp45-hIL10 (SEQ ID NO: 16) 5′agataggaaaatttcgattaagtcatcttacctctt . . . thyA, P1-T7-usp-hIL10

Alternatively, at the 5′ end, the insertion is performed in such a waythat the thyA promoter (Ross, 1990 a) is not included:

(SEQ ID NO: 29) 5′ tctgagaggttattttgggaaatactattgaaccatatcgaggtgtgtggtataatgaagggaattaaaaaagataggaaaatttcatg . . . thyA wild-type(SEQ ID NO: 29) gattaagtcatcttacctctt . . . P1-T7-usp45-hIL10(SEQ ID NO: 14) 5′ tctgagaggttattttgggaaatactagattaagtcatcttacctctt . . . thyA, P1-T7-usp45-hIL10

At the 3′ end, an ACTAGT SpeI restriction site was engineeredimmediately adjacent to the TAA stop codon of the usp45-hIL-10 sequence.This was ligated in a TCTAGA XbaI restriction site, which was engineeredimmediately following the thyA stop codon

(SEQ ID NO: 30) aaaatccgtaac taa ctagt 3′ . . . usp45-hIL10(SEQ ID NO: 31) gatttagcaatttaaattaaattaatctataagtt 3′ . . .thyA-wild-type (SEQ ID NO: 32) tctagaattaatctataagttactga 3′. . . engineered thyA target (SEQ ID NO: 18) aaaatccgtaac taactagaattaatctataagttactga 3′ . . . thyA, usp45-hIL10

These constructs are depicted in FIG. 2. The sequences of pOThy11,pOThy12 pOThy15 and pOThy16 are given by SEQ ID NOs: 23, 24, 25, and 26respectively. The resulting strains are thyA deficient, a mutant not yetdescribed for L. lactis. It is strictly dependent upon the addition ofthymine or thymidine for growth.

The map of the deletion, as well as the PCR analysis of all theisolates/mutants of the present invention, is shown in FIGS. 3A-4B. Thepresence of the thymidylate synthase and the Interleukin 10 (IL-10) genein the wild-type strain and in the independent isolates/mutant wasanalyzed by Southern analysis as shown in FIGS. 5A-6B. The region aroundthe inserted hIL-10 gene was isolated by PCR and the DNA sequence wasverified. The structure is identical to the predicted sequence.

Human Interleukin-10 (hIL-10) production in the mutants was checked bywestern blot analysis and compared with the parental strain, transformedwith pTREX1 as negative control, and the parental strain, transformedwith the IL10-producing plasmid pT1HIL10apxa as a positive control (FIG.7A). The concentration in the culture supernatant was quantified usingELISA. As shown in FIG. 7B, both isolates of the mutant produce acomparable, significant amount of hIL-10, be it far less than thestrain, transformed with the nonintegrative plasmid pT1HIL10apxa. FIGS.8A and 8B further demonstrate that all mutants produce a significantamount of hIL-10.

FIG. 9 shows the production of hIL-10 by the L. lactis strains LL108carrying pOThy11, pOThy12, or pOThy16. Quantification (by ELISA) ofhIL-10 present in the culture supernatant of the indicated strains isshown. The N-terminal protein sequence of the recombinant hIL-10 wasdetermined by Edman degradation and was shown to be identical to thestructure as predicted for the mature, recombinant hIL-10. The proteinshowed full biological activity. LL108 is an L. lactis strain carrying agenomic integration of the repA gene, required for replication of pORI19derived plasmids such as pOThy 11, pOThy12, pOThy15 or pOThy16. Thisstrain was kindly donated by Dr. Jan Kok, University of Groningen, TheNetherlands. The plasmids pOThy11, pOThy12, pOThy15 and pOThy16 carrythe synthetic human IL-10 gene in different promoter configurations (seeFIG. 2), flanked by approximately 1 kB of genomic DNA derived from thethyA locus, upstream and downstream from thyA. These plasmids were usedfor the construction of the genomic integration as described.

The effect of the thymidylate synthase deletion on the growth inthymidine less and thymidine-supplemented media was tested; the resultsare summarized in FIGS. 10 and 11. An absence of thymidine in the mediumstrongly limits the growth of the mutant and even results in a decreaseof colony-forming units after four hours of cultivation. The addition ofthymidine to the medium results in an identical growth curve and amountof colony-forming units, compared to the wild-type strain, indicatingthat the mutant does not affect the growth or viability inthymidine-supplemented medium. FIG. 11 clearly demonstrates that Thy12viability is severely impaired in the absence of thymidine.

FIG. 12 finally shows that L. lactis Thy12 passes through the intestineof the mice at the same speed as MG1363. Loss of viability does notappear to differ between Thy12 and MG1363. Thy12 appears fully dependenton thymidine for growth, indicating that no Thy12 bacteria had taken upa foreign thyA gene.

REFERENCES

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1-20. (canceled)
 21. An isolated thymidylate synthase (thyA) mutant ofLactococcus species comprising an inactive thymidylate synthase gene anda gene or expression unit encoding a heterologous prophylactic and/ortherapeutic molecule.
 22. The isolated thyA mutant of Lactococcusspecies according to claim 21, wherein the inactive thymidylate synthasegene has been inactivated by gene disruption.
 23. The isolated thyAmutant of Lactococcus species according to claim 22, wherein theinactive thymidylate synthase gene has been disrupted by the gene orexpression unit encoding the heterologous prophylactic and/ortherapeutic molecule.
 24. The isolated thyA mutant of Lactococcusspecies according to claim 21, wherein said Lactococcus species isLactococcus lactis.
 25. The isolated thyA mutant of Lactococcus speciesaccording to claim 21, wherein said isolated thyA mutant is transformedwith a transforming plasmid, wherein said transforming plasmid does notcomprise an intact thymidylate synthase gene.
 26. The isolated thyAmutant of Lactococcus species according to claim 21, wherein theheterologous prophylactic and/or therapeutic molecule is interleukin-10.27. A pharmaceutical composition comprising an isolated thymidylatesynthase (thyA) mutant of Lactococcus species, said isolated thyA mutantcomprising an inactive thymidylate synthase gene and a gene orexpression unit encoding a heterologous prophylactic and/or therapeuticmolecule.
 28. The pharmaceutical composition according to claim 27,wherein the inactive thymidylate synthase gene has been inactivated bygene disruption.
 29. The pharmaceutical composition according to claim28, wherein the inactive thymidylate synthase gene has been disrupted bythe gene or expression unit encoding the heterologous prophylacticand/or therapeutic molecule.
 30. The pharmaceutical compositionaccording to claim 27, wherein said Lactococcus species is Lactococcuslactis.
 31. The pharmaceutical composition according to claim 27,wherein said isolated thyA mutant is transformed with a transformingplasmid, wherein said transforming plasmid does not comprise an intactthymidylate synthase gene.
 32. The pharmaceutical composition accordingto claim 27, wherein the heterologous prophylactic and/or therapeuticmolecule is interleukin-10.
 33. A method for delivering a prophylacticand/or therapeutic molecule to a subject, the method comprisingadministering to the subject an isolated thymidylate synthase (thyA)mutant of Lactococcus species comprising an inactive thymidylatesynthase gene and a gene or expression unit encoding the heterologousprophylactic and/or therapeutic molecule.
 34. The method according toclaim 33, wherein the inactive thymidylate synthase gene has beeninactivated by gene disruption.
 35. The method according to claim 34,wherein the inactive thymidylate synthase gene has been disrupted by thegene or expression unit encoding the heterologous prophylactic and/ortherapeutic molecule.
 36. The method according to claim 33, wherein saidLactococcus species is Lactococcus lactis.
 37. The method according toclaim 33, wherein said isolated thyA mutant is transformed with atransforming plasmid, wherein said transforming plasmid does notcomprise an intact thymidylate synthase gene.
 38. The method accordingto claim 33, wherein the heterologous prophylactic and/or therapeuticmolecule is interleukin-10.
 39. A method for treating inflammatory boweldisease in a subject, the method comprising administering to the subjectan isolated thymidylate synthase (thyA) mutant of Lactococcus speciescomprising an inactive thymidylate synthase gene and a gene orexpression unit encoding the heterologous prophylactic and/ortherapeutic molecule.
 40. The method according to claim 39, wherein theinactive thymidylate synthase gene has been inactivated by genedisruption.
 41. The method according to claim 40, wherein the inactivethymidylate synthase gene has been disrupted by the gene or expressionunit encoding the heterologous prophylactic and/or therapeutic molecule.42. The method according to claim 39, wherein said Lactococcus speciesis Lactococcus lactis.
 43. The method according to claim 39, whereinsaid isolated thyA mutant is transformed with a transforming plasmid,wherein said transforming plasmid does not comprise an intactthymidylate synthase gene.
 44. The method according to claim 39, whereinthe heterologous prophylactic and/or therapeutic molecule isinterleukin-10.